Objectives under this grant have been to identify nutritionally important substances required by microorganisms, then to clarify their metabolic role in bacteria. We have paid particular attention to the lactic acid bacteria, and to the nutrients vitamin B6 and pantothenic acid. Our specific objectives for the first years of this grant will include studies of several independent problems as follow: (1) Homogeneous histidine decarboxylase from Lactobacillus 30a contains a pyruvoyl residue and essential -SH group in the active center. Sequence studies are planned to determine the proximity of these two groups in the primary structure; if suitable crystals can be obtained, we will explore collaborative determination of their 3-dimensional structure. Histidine methyl ester inhibits irreversibly by binding at the active site. If partial hydrolysis products can be obtained with this or other active site inhibitors, further mapping of the active site will be possible. Histidine decarboxylase arises from an inactive proenzyme that lacks the pyruvate residue and contains only one species of peptide chain. Activation of the proenzyme involves chain cleavage and conversion of a serine to a pyruvate residue. We will study effects of inhibitors, ionic strength, crude extracts, etc., to determine the nature of this conversion. (2) Studies of active site residues and mechanism of action of tryptophanase from E. coli and of histidinol-P aminotransferase will be continued. (3) We will continue studies of the uptake and transport of vitamin B6 and pantothenic acid by both E. coli and organisms that are auxotrophic for these vitamins. Membrane receptor proteins involved in active transport will be sought. (4) Utilization of vitamin B6 as a carbon source for bacteria involves a flavin-dependent oxygenase that reduces and inserts oxygen into the substrate. Isolation of the crystalline enzyme, redetermination of the extent of O2-incorporation, its iron content, and the specificity of its FAD requirement should clarify the nature and mechanism of action of this unusual oxygenase.